Modified ether glyceroglycolipids

ABSTRACT

This invention provides lipids having: 1) a glycerol backbone; 2) a hydrocarbon chain, preferably saturated and containing 16 or 18 carbon atoms, attached to C-1 of the backbone by an ether linkage, 3) a methyl group attached to C-2 of the backbone, preferably by an ether linkage; and, 4) a sugar attached to C-3 of the glycerol backbone in either the alpha or beta anomeric configuration, the sugar being altered by modification of, or substitution for, one or more of its hydroxyl groups. Also provided herein are ether-lipid-containing compositions, as well as methods of administering such compositions to animals, for example, those afflicted with cancers, as well as various other diseases and disorders.

This application is a continuation of our U.S. application Ser. No.08/722,881, filed Sep. 26, 1996, now U.S. Pat. No. 6,153,736, whichclaims the benefit of U.S. Provisional Application No. 60/004,398, filedSep. 27, 1995, and now abandoned.

1. Filed of the Invention

This invention is directed to modified ether glyceroglycolipids,compositions containing these compounds, and to the therapeuticadministration of these compounds and compositions to animals, includingthose afflicted with cancers, as well as various other diseases anddisorders.

2. Background of the Invention

Etherlipids are amphipathic lipids with ether linkages connecting theirhydrocarbons with their molecular backbones, and are synthetic analogsof platelet activating factor (“PAF”;1-O-2-acetyl-sn-glycero-3-phosphocholine). PAF is an effector believedto be involved in a variety of physiological processes, such asinflammation, immune responses and allergic reactions.

Etherlipids can accumulate in cell membranes, following which the lipidsmay affect the cells in a number of ways. Cell membrane accumulation canlead to disturbance of membrane lipid organization by a detergent-likeactivity of etherlipids; membrane structure, and hence, cell stability,can be disrupted by this activity. Phospholipid metabolism can also bedisrupted, as the activities of several of the enzymes involved, e.g.,CTP: phosphocholine cytidyl transterase, diacylglycerol kinase,sodium/potassium adenosine triphosphate phosphatase, acyl transferases,lysophospholipase, and phospholipases C and D, are inhibited in thepresence of etherlipids. Etherlipids can also affect transmembranesignaling pathways, nutrient uptake, cellular differentiation andapoptosis.

Moreover, etherlipids are believed to be cytotoxic to cancer cells, andhave been shown to be effective anticancer agents in animals (see, forexample, Lohmeyer and Bittman, 1994; Lu et al. (1994a); Lu et al.(1994b); Dietzfelbinger et al. (1993); Zeisig et al. (1993); Berdel(1991); Workman (1991); Workman et al. (1991); Bazill and Dexter (1990);Berdel (1990); Guivisdalsky et al. (1990a); Guivisdalsky et al. (1990b);Powis et al. (1990); Layton et al. (1980): Great Britain Patent No.1,583,661; U.S. Pat. No. 3,752,886). However, etherlipids are generallynot toxic to normal cells. Ether lipids' ability to act selectively oncancer cells is believed to be due to the cancer cells' lack of thealkyl cleavage enzymes necessary for hydrolysis of the lipids; theresulting intracellular lipid accumulation can disrupt the cells'functioning in a variety of ways. Normal cells typically possess theseenzymes, and hence, to prevent their intracellular accumulation.

However, not all normal cells contain sufficient levels of alkylcleavage enzymes to prevent intracellular ether lipid accumulation;cells which do not possess the requisite levels of the enzymes can besubject to the same disruptive effects of ether lipid action as arecancer cells. Red blood cells, for example, lack the requisite alkylcleavage enzymes, and hence, are also subject to a detergent-likeactivity of ether lipids. Hemolysis which results from exposure of thesecells to ether lipidis having detergent-like activity can be a majordrawback to therapeutic use of the ether lipids (see, for example,Houlihan et al., 1995).

A number of different approaches are potentially available fordecreasing or eliminating such drug-induced toxicity. One such approachis to incorporate the drugs into lipid-based carriers, e.g., liposomes.Such carriers can buffer drug toxicity, for example, by sequestering thedrug in the carrier such that the drug is unavailable for inducingtoxicity. Lipid carriers ran also buffer drug-induced toxicity byinteracting with the drug such that the drug is then itself unable tointeract with the cellular targets through which it exerts its cytotoxiceffects. The carriers also maintain the ability of the drugs to betherapeutically effective when released therefrom, e.g., when thecarriers are broken down in the vicinity of tumors.

This invention provides etherdipids in which the lipids' phosphate-basedheadgroups have been replaced with sugar moieties, the sugars themselveshaving been modified by substitution of one or more of their hydroxylgroups; applicants have found that such modification of etherlipidsaffords the modified etherlipids beneficial anticancer activity. Certainetherlipid analogues have been mentioned in the art, including O- andS-linked glucose and maltose substitutions of edelfosine'sphosphoryicholine group. However, none of these analogues contain sugarsmodified by replacement of one or more hydroxyl groups.

SUMMARY OF THE INVENTION

Etherlipids of this invention are amphipathic lipid molecules comprisinga polyol backbone, a hydrocarbon chain, a methyl group and a modifiedsugar moiety. The ether lipids have the following structural formula:

The hydrocarbon, attached to the polyol by way of an ether linkage, isdesignated herein as “R¹” and is a group having the formula Y¹Y²,wherein Y¹ is the group—(CH₂)_(n1)((CH═CH)_(n2)(CH₂)_(n3)(CH═CH)_(n4)(CH₂)_(n5)(CH═CH)_(n6)(CH₂₎_(n7)(CH═CH)_(n8)(CH₂)_(n9)— and Y² is CH₃, CO₂H or OH. Preferably, thehydrocarbon is saturated and Y¹ is —C(O)(CH₂)_(n1); Y² is preferablyCH₃. Most preferably, presently, the hydrocarbon is —C(O)(CH₂)₁₆CH₃. Themethyl group is attached to the polyol by way of a linkage, designatedherein as “R² which is O, S, NH, or —NHC(O)—. Most preferably, R² is O;accordingly, this invention's glycerol-based etherlipids preferably havea methoxy group at the sn-2 position. The modified sugar attached to thepolyol, and designated herein as “R³”, has the following formula:

wherein X², X³, X⁴, X⁵, X⁶ and X⁷ are either H, OH or a substitution forone of these groups. No more than two of X², X³, X⁴, X⁵ X⁶ and X⁷ areOH, and no more than two of X²/X³, X⁴/X⁵ and X⁶/X⁷ are H/OH or OH/H,when X⁸ is CH₂OH, i.e., when a group other than the OH at C-6 ismodified. No more than three of X², X³, X⁴, X⁵X⁶ and X⁷ are OH, and nomore than three of X²/X³, X⁴/X⁵ and X⁶/X⁷ are H/OH or OH/H, when X⁸ isthe group —COOX¹⁰.

Etherlipids are known to be effective anticancer agents, and can alsoexert beneficial therapeutic activity against a variety of otherdiseases and disorders, such as those characterized by inflammation andby microbial infection. Moreover, etherlipids are relatively inactivetowards most normal cells. This ability of etherlipids to be selectivelycytotoxic to particular target cells is believed to be due to the targetcells' lack of the alkyl cleavage enzymes required for hydrolysis of thelipids: normal cells typically possess sufficient levels of theseenzymes to prevent intracellular ethedipid accumulation, while cancercells generally do not. However, some normal cells, e.g., red bloodcells, do not possess the requisite alkyl cleavage enzymes in sufficientquantities to prevent etherlipids from accumulating therein to toxiclevels; accordingly, ethehipids can be cytotoxic to such cells as well.Etherlipids are incorporated into lipid-based carriers herein such thatthe etherlipids cannot then be exchanged into cell membranes.Nevertheless, the etherlipids are maintained in a therapeuticallyeffective form within the carrier, and when released therefrom, can actagainst their intended targets.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Reaction scheme for the synthesis of2′-deoxy-β-D-arabinopyranosyl and 2-O-methyl-β-D-glucopyranosyl modifiedether glyceroglycolipids. a: Trimethylsilyltrifluoromethanesulfonate/dichloromethane/molecular sieves 3angstroms/minus 78 deg. C./10 min. b: NH₃—MeOH. c: NaH/DMF/Mel. d:Pd—C/1:1 THF—AcOH, e: CS₂/NaH/imidazole/Mel. f: di-N-butyltinoxide/toluene.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides an etherlipid having the formula:

wherein R¹ is the group Y¹Y². Y¹ is a group having the formula—(CH₂)_(n1)(CH═CH)_(n2)(CH₂)_(n3)(CH═CH)_(n4)(CH₂)_(n5)(CH═CH)_(n6)(CH₂)_(n7)(CH═CH)_(n8)(CH₂)_(n9)—.The etherlipid is thus a glycerol-based lipid having a hydrocarbon chainat the sn-1 position, linked to the glycerol backbone by an etherlinkage.

The sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer if from 3 to 23,n1 is equal to zero or an integer of from 1 to 23, n3 is equal to zeroor an integer of from 1 to 20, n5 is equal to zero or an integer of from1 to 17, n7 is equal to zero or an integer of from 1 to 14 and n9 isequal to zero or an integer of from 1 to 11. Each of n2, n4, n6 and 8 isindependently zero or 1. The hydrocarbon is preferably an unsaturatedalkyl chain; accordingly, n2, n4, n6 and n8 are each preferably equal tozero, n3, n5, n7 and n9 are each also equal to zero, and Y¹ is the group—C(O)(CH₂)_(n1). Alternatively, Y¹ can be unsaturated, that is, it canhave one or more double bonds; accordingly, at least one of n2, n4, n6and n8 is then equal to 1. For example, when the unsaturated hydrocarbonhas one double bond n2 is equal to 1, n4, n6 and n8 each then beingequal to zero, and Y¹ is then —C(O)(CH₂)_(n1)CH═CH(CH₂)_(n3).

Y² is CH₃, CO₂H or OH, and is preferably CH₃; accordingly, R¹ ispreferably the group —C(O)(CH₂)_(n1)CH₃. More preferably, R¹ is—C(O)(CH₂)₁₇CH₃.

Etherlipids of this invention also comprise a methyl group, attached tothe glycerol backbone by way of a linkage, designated herein as “R²”,that is O, S, NH, —NHC(O)—or —OC(O)—. Preferably, R² is O; accordingly,this invention's glycerol-based etherlipids preferably have a methoxygroup at the sn-2 position.

Sugars linked to the third carbon of the glycerol backbone have thefollowing formula:

The sugars can be in either the alpha or beta anomeric forms.

Sugars ordinarily have X²/X³, X⁴/X⁵ and X⁶/X⁷ pairs wherein one memberis H and the other is OH. Glucose, for example, has X² being H and X³OH, X⁴ being OH and X⁵ H, while X⁶ is H and X⁷ is OH; in mannose, X² isOH and X³ is H, X⁴ is OH and X⁵ is H, while X⁶ is H and X⁷ is OH. X⁸ istypically CH₂OH in such sugars. This invention provides etherlipidswhose headgroups are sugar moieties modified by alteration of, orsubstitution for, one or more of the sugar hydroxyl groups. No more thantwo of X², X³ X⁴, X⁵ X⁶ and X⁷ are OH's and no more than two of X²/X³,X⁴/X⁵ and X⁶/X⁷ are H/OH or OH/H when X⁸ is CH₂OH, i.e., when a groupother than the OH at C-6 is modified. No more than three of X², X³, X⁴,X⁵ X⁶ and X⁷ are OH's, and no more than three of X²/X³, X⁴/X⁵ and X⁶/X⁷are H/OH or OH/H when X⁸is the group —COOX¹⁰.

Modifications to sugar molecules according to the practice of thisinvention are any atom or group of atoms which: 1) can be modified from,or substituted for, a sugar hydroxyl group; and, 2) enhance the cellgrowth inhibitory activity of a modified sugar-containing etherlipid incomparison to the growth inhibitory activity of the corresponding lipidhaving the same sugar residue at the sn-3 position, wherein the sugar isnot modified at the hydroxyl group. Such modifications include, withoutlimitation, converting a sugar's OH group to H, NH₂, NHCH₃, NH(CH₃)₂,OCH₃, NHC(O)CH₃, F, Cl, Br, I, —OP(O)₃ ³⁻ and —OSO₃ ²⁻. Counterionspresent when the modified sugar is a salt form are those ions typicallyused in connection with the groups, e.g., phosphate and sulfate, withwhich the sugar is modified.

Sugar molecule hydroxyl groups can be modified as described herein,using techniques well known to ordinarily skilled artisans given theteachings of this invention. Comparisons of anticancer activitiesbetween different compounds can be accomplished by means also well knownto ordinarily skilled artisans given the teachings of this invention.These include, for example, in vitro growth inhibition assays such asthose described in Example 18 hereinbelow. Briefly, cells, such ascancer cells, are grown in cultures and the compounds in question areadded to the cultures; the concentrations of the compounds required toachieve a certain percentage, e.g., 5%, 10% or 50%, of growth inhibitionin the cultures (in comparison to control cultures) are then determinedand compared. Compounds which acheive the same level of growthinhibition in a culture at a lower concentration are more effectivegrowth inhibitory agents. Alternatively, an etherlipid can be tested invivo for anticancer activity, for example, by first establishing tumorsin suitable test animals, e.g., immune-deficient mice, administering theetherlipid to the animals and then measuring tumor growth inhibition inthe animals and their survival rates. Cells suitable for such in vitroor in vivo testing include, without limitation: murine P388 leukemia,B16 melanoma and Lewis lung cancer cells; human MCF7, ovarian OVCAR-3and A549 lung cancer cells, as well as other cells generally accepted inthe art for such testing.

The sugar can be modifed at any of its OH groups, which can bedesignated herein as X², X³, X⁴, X⁵, X⁶ or X⁷; the sugar can also bemodified at the OH group of X⁸ which, unmodified, is CH₂OH. Each of X²and X³ can be unaltered from the parent sugar so long as at least one ofX⁴, X⁵, X⁶, X⁷ or X⁸ is then altered; one of X² and X³ is then H whilethe other is OH. Alternatively, the OH group at X² or X³ can be alteredas described herein, to give a modified sugar-containing etherlipid; X²and X³ can then, for example, be H, NH₂, NHCH₃, NH(CH₃)₂, OCH₃,NHC(O)CH₃, F, or Cl. Each of X⁴ and X⁵ can be unaltered from the parentsugar, one then being H while the other is OH; alternatively, thehydroxyl group at X⁴ or X⁵ can be altered to give, at X⁴ or X⁵ NH₂,NHCH₃ or N(CH₃)₂, —OPO₃ ³⁻ or —OSO₃ ²⁻; these include withoutlimitation, sodium and potassium ions, amongst others. Each of X⁶ and X⁷can also be unaltered. When the sugar is a monosaccharide, one of X⁶ andX⁷ is then H, while the other is OH; alternatively, when the sugar is adisaccharide, one of X⁶ and X⁷ is H while the other is a group havingthe formula —OX⁹, wherein X⁹ is an additional sugar molecule, that is, atetrose, pentose, a hexose or heptose sugar, linked through an oxygenatom at X⁶ or X⁷. Disaccharides have the additional sugar linked throughan oxygen at X⁶ or through an oxygen at X⁷. One or more hydroxyl groupson the additional sugar can also be modified according to the practiceof this invention. X⁸ is CH₂OH when the sugar is unmodified at the C-6position, or a group having the formula —OC(O)X¹⁰ when the sugar ismodified at this position, wherein X¹⁰ is H, CH₃ or a group also havingthe formula Y¹Y². In preferred embodiments of this invention, X¹ is O,X⁴ is OH, X⁵ is H, X⁶ is H, X⁷ is OH and X⁸ is CH₂OH. Preferably, whenX² is H, X³ is H, NH₂ or —OCH₃, or when X³is H, X² is H or —OCH₃.

The etherlipids of this invention can be prepared by a number of meansreadily practiced by ordinarily skilled artisans given the teachings ofthis invention for modifying specific groups on sugar molecules.Generally, ethedipid starting material, typically the form of theetherlipid having a phosphorylcholine group at the third position of theglycerol backbone, is glycosylated using a suitable and availableglucose donor (which can also be prepared as described below). Sugar OHgroups are then modified by known means, typically involvingprotection/deprotection of unsubstituted groups, to give the desiredfunctional group substitution.

For example, 1-O-hexadecyl-2-O-methyl-sn-glycerol, synthesized fromD-mannitol (see Baver et al., 1991), or by the Lewis acid-catalyzed(BF₃.Et₂O) regioselective ring opening of (R)-glycidyl arenesulfonateswith 1-hexadecanol (see, Guivisdalsky et al., 1991), can be glycosylatedto give 1-O-hexadecyl-3-O-protected-sn-glycerol. This 3-O-sn-protectedglycerol can be methylated, for example, with diazomethane in thepresence of SiO₂: followed by deprotection, to give1-O-hexadecyl-2-O-methyl-sn-glycerol. Alternatively, ordinarily skilledartisans can readily follow a synthetic scheme based, for example, uponasymmetric dihydroxylation of allyl p-methoxyphenyl ether using a chiralphthalazine ligand, such as AD-mix-alpha in a mixture oftert-butanol-water at 0 deg. C., giving3-O-(p-methoxyphenyl)-sn-glycerol (I; see Vilcheze and Bittman, 1994 andByun et al., 1994). Selective monoalkylation of I with1-bromohexadecane, in DMF, via 1,2-O-stannylidene in the presence of CsF(see Nagashima et al., 1987) gives a mixture of sn-1-O-hexadecyl (II)and sn-2-O-hexadecyl glycerols. Following chromatographic separation ofthese two isomers, II is methylated by treatment with Mel-NaH-DMF; the3-O-(p-methoxyphenyl) function is then removed with ammonium cerium (IV)nitrate, in aqueous acetonitrile, to give1-O-hexadecyl-2-O-methyl-sn-glycerol.

Synthesis of analogs with either 2′-deoxy or 2′-O-alkyl functions onmonosaccharide residues generally requires that the C-3, C-4 and C-6protecting groups of the glycosyl donor allow for preferentialdeprotection of the C-2 glycoside protecting group, resist2′-O-alkylation and resist deoxygenation.2-O-Acetyl-3,4,6-tri-O-benzyl-α,β-D-glucopyranosyl trichloroacetimidateand 2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-mannopyranosyltrichloroacetimidate meet these requirements and can be made, forexample, from their respective benzylated 1,2-orthoesters. Briefly, forexample, to synthesize2′-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-glucopyranosyltrichloroacetimidate, the benzylated 1,2-orthoester is acetolized inglacial acetic acid (see Boren et al., 1973, Lemieux et al., 1956 andTrumtel et al., 1989) to give 1′,2′-trans-di-O-acetate, the 1-O-acetatefunction of which is then removed selectively with hydrazine acetate inDMF to give the hemiacetal quantitatively, after aqueous workup. Ananomeric mixture of 2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-glucopyranosyltrichloroacetimidate isomers is obtained by treating the hemiacetal withtrichloroacetonitrile-potassium carbonate in dichloromethane, and wasthen purified by flash chromatography. Synthesis of2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-mannopyranosyl trichloroacetimidateinvolves hydrolysis of the benzylated 1,2-orthoester with acetic acid atroom temperature, generally about 25 deg. C., for about 6 hours,followed by treatment of the resulting hemiacetal withtrichloroacetonitrile-potassium carbonate in dichloromethane.

Functionalization of the C-2′ position of the monosaccharide attached tothe etherlipid can, for example, be carried out according to thereaction scheme depicted in FIG. 1 and described below. Briefly, the2-O-acetyl functions of1-O-hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-β-D-glucopyranosyl)-sn-glyceroland1-O-hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-α-D-mannopyranosyl)-sn-glycerolare quantitatively removed by NH₃/MeOH aminolysis, followed bymethylation of the 2′ hydroxy group with NaH-DMF-Mel to give1-O-hexadecyl-2-O-methyl-3-O-(2′-O-methyl-3′,4′,6′-tri-O-benzyl-β-D-glucopyransoyl)-sn-glycerolor1-O-hexadecyl-2-O-methyl-3-O-2-O-methyl-3′,4′,6′-tri-O-benzyl-α-D-mannopyransoyl)-sn-glycerol,respectively. The O-benzyl protecting groups are then removed in 1:1THF-HOAc using Pd-C, under a balloon pressure of hydrogen, to give2-O-methyl-β-D-glycopyransoyl and 2-O-methyl-α-D-mannopyransoyletherlipids. Low molecular weight impurities can be removed from suchcompounds by filtration, for example, through lipophilic Sephadex LH-20using methanol.

Further useful synthetic techniques include radical xanthate reduction,which is a procedure commonly used to deoxygenate carbinols. Alcoholscan be converted to the corresponding xanthates, for example, bytreatment in tetrahydrofuran (THF) with sodium hydride, carbon disulfideand a catalytic amount of imidazole, followed by reaction with Mel. Thexanthates can the be converted to the corresponding 2′-deoxy-glycosidesby radical reduction with dibutyltin oxide (see Barton et al., 1975; andHartwig, 1983) in the presence of azobisisobutyronitrile (AIBN);confirmation of the reduction can be made by NMR spectroscopy. Thecontents of the above cited disclosures of reactions and syntheticschemes for the modification of sugar molecule OH groups areincorporated herein by reference.

Also provided herein is a composition comprising an etherlipid of thisinvention; the composition preferably also comprises a pharmaceuticallyacceptable medium, which are media generally intended for use inconnection with the administration of active ingredients to animals, andare formulated according to a number of factors well within the purviewof the ordinarily skilled artisan to determine and account for. Theseinclude, without limitation: the particular active ingredient used, itsconcentration, stability and intended bioavailability; the disease,disorder or condition being treated with the composition; the subject,its age, size and general condition; and the composition's intendedroute of administration (see, for example, J. G. Naim, in: Remington'sPharmaceutical Science (A. Gennaro, ed.), Mack Publishing Co., Easton,Pa., (1985), pp. 1492-1517, the contents of which are incorporatedherein by reference). Pharmaceutically acceptable media include, withoutlimitation: solids, such as pills. capsules and tablets; gels;excipients; and aqueous or nonaqueous solutions. Typicalpharmaceutically acceptable media used in parenteral drug administrationinclude, for example, D5W, an aqueous solution containing 5% weight byvolume of dextrose, and physiological saline.

Etherlipid-containing compositions provided herein preferably alsocomprise a lipid carrier with which the etherdipid is associated. “Lipidcariers” are hydrophobic or amphipathic molecules suitable foradministration to animals, and include, without limitation: fatty acids,phospholipids, micelles, lipoproteins, nonliposomal lipid-basedcomplexes and liposomes. Preferably, the lipid carrier is a liposome,which comprises one or more bilayers of lipid molecules, each bilayerencompassing an aqueous compartment. The amphipathic lipid moleculeswhich make up lipid bilayers comprise polar (hydrophilic) headgroups andnonpolar (hydrophobic) hydrocarbon chains. The polar groups can bephosphate-, sulfate- or nitrogen-based groups, but are preferablyphosphate groups such as phosphorylcholine, phosphorylethanolamine,phosphorylserine, phosphorylglycerol or phosphorylinositiol. Thehydrocarbons generally comprise from 12 to 24 carbon atoms, and can besaturated (e.g., myristic, lauric, palmitic, or stearic acid), orunsaturated (e.g., oleic, linolenic or arachidonic acid). Liposomalbilayers can also include sterols, such as cholesterol, other lipids andnonlipid molecules.

“Association” between an etherlipid and a lipid carrier is by way of anumber of influences, such as Van der Waals forces, generally known tooperate between hydrophobic molecules in an aqueous environment. Meansof determining the stability of such associations, for example, bydetermining the percentage of etherlipid recoverable with phosphorouswhen the lipid carrier comprises a phospholipid, are well known to, andreadily practiced by, ordinarily skilled artisans given the teachings ofthis invention.

Lipid carrier-based formulations can enhance the therapeutic index of anassociated etherlipid, by buffering the lipid's toxicity-causingpotential while maintaining or enhancing its therapeutic efficacy, forexample, by increasing the amount of the etherlipid-lipid carrierassociation reaching the intended site of therapeutic action. Preferredmeans for doing so include increasing the length of time in which theetherlipid-lipid carrier association remains in the circulation ofanimals to which it has been administered. In the case of cancertreatment, for example, increasing circulatory half-life allows more ofthe administered material to reach tumors, which tend to have anincreased amount of vasculature in comparison to surrounding tissue;this vasculature also tends to be more leaky than that found in healthytissue, meaning that etherlipid-lipid carrier associations can readilyreach leak out into surrounding tumor tissue.

Preferred means for enhancing etherlipid-lipid carrier circulation is byincorporating a “headgroup-modified lipid” into the lipid carrier.Headgroup-modified lipids, e.g., phosphatidylethanolamines (“PE's”),generally comprise polar groups derivatized by attachment thereto of amoiety, e.g., dicarboxylic acids such as succinic and glutaric acids,which can inhibit the binding of serum proteins to the carriers so thatthe pharmacokinetic behavior of the carriers is altered (see, e.g.,Blume et al., Biochim. Biophys. Acta. 1149:180 (1993); Gabizon et al.,Pharm. Res. 10(5):703 (1993); Park et al. Biochim. Biophys Acta.1108:257 (1992); Woodle et al., U.S. Pat. No.5,013,556; and Allen etal., U.S. Pat. Nos. 4,837,028 and 4,920,016, the contens of which areincorporated herein by reference). The amount of the headgroup-modifiedlipid incorporated into the lipid carrier generally depends upon anumber of factors well known to the ordinarily skilled artisan, orwithin his purview to determine without undue experimentation, given theteachings of this invention. These include, but are not limited to: thetype of lipid and the type of headgroup modification; the type and sizeof the carrier; and the intended therapeutic use of the formulation.Typically, from about 5 to about 20 mole percent of the lipid in aheadgroup-modified lipid-containing lipid carrier is headgroup-modifiedlipid.

Further provided herein is a method of administering an etherlipid to ananimal, which comprises administering an etherlipid-containingcomposition of this invention to the animal. The animal is preferably ahuman, and administration is preferably intravenous, but can also be byany other means generally accepted for administration of therapeuticagents to animals. Etherlipid-containing compositions provided hereincan be administered prophylactically or therapeutically to animals atrisk of, or afflicted with, various diseases and disorders whichinclude, without limitation, cancerous, inflammatory and infectiousconditions.

Cancers, e.g., brain, breast, lung, colon, ovarian, prostate, liver orstomach cancers, and carcinomas, sarcomas and melanomas, can be treatedwith the etherlipid-containing compositions of this invention. Thecompositions are particularly useful for the treatment of drug-resistantcancers, i.e., forms of a cancer resistant to one or more drugs, e.g.,adriamycin, commonly used to treat the cancer. Preferably, compositionsused to treat cancers comprise, in addition to an etherlipid, a lipidcarrier, more preferably, a liposome. Most preferably, the liposome is aunilamellar liposome having an average diameter of from about 100 nm toabout 200 nm.

Animals treated for cancers according to the practice of this inventionare given an anticancer effective amount of an etherlipid. “Anticancereffective amounts” of an etherlipid are any amount of the etherlipideffective to ameliorate, lessen, inhibit or prevent the establishment,growth, metastasis, or invasion of a cancer. Generally, the anticancereffective amount of the etherlipid is at least about 0.1 mg of theetherlipid per kg of body weight of the animal to which theetherlipid-containing composition is administered. Typically, theanticancer effective amount of the etherlipid is from about 0.1 mg perkg of body weight of the animal to about 1000 mg per kg; preferably, theanticancer effective amount is from about 1 mg of the lipid per kg toabout 200 mg per kg. Within these ranges, etherlipid doses are chosen inaccordance with a number of factors, e.g., the age, size and generalcondition of the subject, the cancer being treated and the intendedroute of administration of the lipid, well known to, and readilypracticed by, ordinarily skilled artisans given the teachings of thisinvention.

Etherlipid treatment can follow a variety of accepted chemotherapeuticregimens, and can include administration of an anticancer effectiveamount in segments over a suitable period of time, or repeatedadministrations of an anticancer effective amount, each dosing beingseparated by a suitable period of time. Additional bioactive agents,i.e., bioactive agents in addition to the etherlipid, can beadministered to the animal in accordance with the practice of thisinvention, either concurrently with, or separately from etherlipidadministration, and either as a component of the same, or a different,composition. “Bioactive agents” are compounds or compositions of matterhaving biological activity on animal cells in vitro or when administeredto an animal; bioactive agents can have therapeutic and/or diagnosticactivity. Such agents include, but are not limited to, antimicrobial,anti-inflammatory and anticancer agents, as well as radioactive agents,enzymes isotopes and dyes.

This invention will be better understood from the following Examples.However, those of ordinary skill in the art will readily understand thatthese examples are merely illustrative of the invention as defined inthe claims which follow thereafter.

EXAMPLES Example 1

Materials and Methods

Silica gel GF TLC plates of 0.25-mm thickness (Analtech, Newark, Del.)were used to monitor reactions, with visualization by charring using 10%sulfuric acid in ethanol and/or short wavelength ultraviolet light.Flash chromatography was carried out with silica gel 60 (230-400 ASTMmesh) of E. Merck (purchased from Aldrich), isocratically unlessotherwise stated. ¹H NMR spectra were recorded on IBM-Bruker WP-200 andAMX400 spectrometers, at 200 and 400.13 MHz, respectively, in CDCl₃solutions—chemical shifts are in parts per million fromtetramethylsilane as the internal standard; ¹³C-NMR spectra wererecorded at 75 MHz and 100.57 MHz, respectively—¹³C chemical shifts aregiven by assigning 77.0 ppm for the central line of CDCl₃. Opticalrotations were measured at 20±2 deg. with a JASCO DIP-140 digitalpolarimeter, in a cell of 1-dm path length; 1% solutions in chloroformwere used, unless otherwise stated. The melting points are uncorrected.

Trity chloride was obtained from Aldrich. Zinc chloride was obtainedfrom Fluka. Dichloromethane was dried over P₂O₅ and distilled justbefore use, or refluxed over calcium hydride and distilled under apositive nitrogen pressure before use; tetrahydrofuran was refluxed oversodium benzophenone ketone and distilled before use; methanol wasrefluxed over Mg(OMe)₂ and distilled before use; toluene was distilledand then redistilled from calcium hydride before use; anhydrous N,N-dimethyl formamide (DMF) was acquired from Janssen Chimica. Solidsynthons were dried under vacuum (0.2 mm Hg), and all reactions werecarried out under dry nitrogen using air-sensitive glassware (greaselessvacuum/gas manifold). Nitrogen gas was dried through a drying tower ofgranulous anhydrous calcium chloride. Molecular sieves of 3 angstromswere dried at 150 deg. C., under vacuum, over P₂O₅ for 12 hours, andstored under vacuum over P₂O₅.

Human epithelial cancer cell lines were grown from frozen stocksoriginally obtained from the ATCC, in media commonly used for growingthese cells in culture. For example, A549 cells (non-small cell lungadenocarcinoma) were cultured in Ham's F-12 medium, T84 cells (coloncarcinoma) were cultured in a 1:1 mixture of F-12 and DMEM while MCF-7(breast adenocarcinoma) and A427 (large cell lung carcinoma) cells werecultured in DMEM. The media were supplemented with 10% fetal bovineserum, penicillin (50 U/ml), streptomycin (50 micogram/ml) and fungizone(0.5 microgram/ml). OVCAR-3 cells (ovarian adenocarcinoma) were culturedin RPMI 1640 medium supplemented with 20% FBS and 10 microgram/mlinsulin.

Cells were subcultured into 24-well plates and the cell number wasmonitored daily. When the cells were in log phase, the media wasreplaced with one containing the required drug concentration, and thecells were incubated for 72 h. The increase in cell numbers relative tocontrol wells (without any drug) was determined after the incubation.Stock solutions of the drugs (30 micromolar) were prepared in ethanoland stored at −20 deg. C. Etherlipid solutions (30 micromolar) in theappropriate media were prepared fresh on the day of the experiment andserially diluted to give the required concentrations. The finalconcentration of ethanol in all wells was 0.1% (v/v).

Example 2

Synthesis of 1-O-hexadecyl-2-O-methyl-sn-gylcerol

To a solution of1-O-hexadecyl-2-O-methyl-3-O-p-methoxyphenyl)-sn-glycerol (1.0 g, 2.3mmol) in 4:1 acetonitrile-water (21 ml) was added ammonium cerium(IV)nitrate (2.9 g, 5.5 mmol) at 0 deg. C., with vigorous stirring. Theresulting mixture was warmed to room temperature, and stirred for onehour, following which TLC (4:1 hexane:ethyl acetate) showed completeconversion of the starting material to1-O-hexadecyl-2-O-methyl-sn-glycerol. The reaction was quenched byaddition of 1.0 g sodium sulfite.

The resulting mixture was diluted with ethyl acetate, and the organicsolution was washed with water, brine, and dried with sodium sulfate; itwas then filtered, and the filtrate evaporated. The residue was purifiedby column chromatography (6:1 hexane:ethyl acetate) to give1-O-hexadecyl-2-O-methyl-sn-glycerol (0.853 g, 94%) as a low meltingpoint white solid. [α]_(D)-9.5 deg.

Example 3

Synthesis of1-O-hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)-sn-glycerol

To a solution of2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-D-glucopyranosyl chloride(219.4 mg, 0.6 mmol), 1-O-hexadecy-2-O-methyl-sn-glycerol (100 mg, 0.3mmol), and trityl chloride (83.6 mg, 0.3 mmol) were added 41.2 mg (0.3mmol) of zinc chloride in dry dichloromethane (5 ml; see Kumar et al.,1994). The reaction mixture was stirred for 4 h at room temperature, andreaction progress was monitored by TLC analysis, in ethyl acetate. Thereaction mixture was diluted with ethyl acetate (50 ml), washed with 5%aqueous sodium bicarbonate solution, washed with water, dried oversodium sulfate, and concentrated under reduced pressure. The residue waspurified by flash chromatography (elution with hexane/ethyl acetate1:1), giving 140 mg (70%) of1-O-hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)-sn-glycerolas a white solid; R_(f). 0.54 (ethyl acetate); [α]_(D)25 −1.31 deg.(c5.6, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ0.80 (t, 3H, J=5.96 Hz, CH₃),1.25 (br, 26H, (CH₂)₁₃CH₃), 1.45 (2H, OCH₂CH₂), 1.87, 1.95, 2.01 (s,12H, OAc, and NAc), 3.32-3.41 (m, 8H, with a singlet at d 3.36,CH₂OCH₂C₁₅H₃₁, CH₃OCH), 3.63 (m, 3H, H-5 and OCH₂), 3.81 (m, 1H, H-2),4.02 (dd, 1H, H-6a), 4.08 (dd, 1H, J=4.57 Hz, H-6b), 4.60 (d, 1H, J=8.34Hz, H-1), 5.05 (t, 1H, J=9.50 Hz, H-4), 5.17 (t, 1H, J=9.83 Hz, H-3),5.84 (d, 1H, J=8.51 Hz, NH).

Example 4

Synthesis of1-O-hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-β-D-glucopyranosyl)-sn-glycerol

1-O-Hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)-sn-glycerol(140 mg, 0.21 mmol), prepared aacording to the procedures of Example 3as described above, was dissolved in 3 ml of 0.25 N methanolic KOH, andthe mixture was stirred for 2 h at room temperature. The reactionmixture was neutralized with saturated aqueous ammonium chloridesolution and extracted with chloroform (10 ml). The chloroform layer wasdried over MgSO₄ and concentrated under reduced pressure and the residuewas purified by flash chromatography (elution with 10% methanol inchloroform), giving 109 mg (96%) of1-O-hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-β-D-glucopyranosyl)-sn-gycerolas a white solid; mp 150-153 deg. C.; R_(f) 0.56 (CHCl₃—CH₃OH 4:1);[α]_(D) ²⁵ −2.26 deg. C. (c 5.25, CHCl₃—CH₃OH); ¹H NMR (200 MHz, CDCl₃and a few drops of CD₃OD) δ0.80 (t, 3H, J =6.33 Hz, CH₃), 1.25 (br, 26H,(CH₂)₁₃CH₃), 1.56 (2H, OCH₂CH₂), 2.01 (s, 3H, NAc), 3.23-3.83 (m, 19H,with a singlet at δ3.45, CH₂OCH₂C₁₅H₃₁, CH₃OCH, OCH₂ and —CHO-'s ofsugar moiety), 4.43 (d, 1H, J=6.84 Hz, H-1), 7.51 (d, 1H, J=8.51 Hz,NH).

Example 5

Synthesis of1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-D-glucoryranosyl)-sn-glycerol

1-O-Hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-β-D-glucopyranosyl)-sn-glycerol(24 mg, 45.3 μmol), prepared as described above, was dissolved in 2 mlof 2 N ethanolic KOH. The mixture refluxed for 4 h, cooled, and thenneutralized with saturated aqueous ammonium chloride solution; theproduct was extracted with chloroform. The chloroform layer was driedover MgSO₄ and concentrated under reduced pressure; the residue waspurified by flash chromatography (elution with 20% methanol inchloroform), giving 18 mg (82%) of1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2-deoxy-β-D-glucopyranosyl)-sn-glycerolas a white solid. R_(f) 0.28 (CHCl₃—CH₃OH 4:1); [α]_(D) ²⁵ −14.40 deg.(c 7.5, CHCl₃/CH₃OH 1:1 (v/v); ¹H NMR (200 MHz, CDCl₃ and a few drops ofCD₃OD) δ0.85 (t, 3H, J=6.34 Hz, CH₃), 1.23 (br, 26H, (CH₂)₁₃CH₃), 1.53(2H, OCH₂CH₂), 3.40-3.91 (m, 20H, with a singlet at d 3.45,CH₂OCH₂C₁₅H₃₁, CH₃OCH, OCH₂, and —CHO-'s of sugar moiety) 4.82 (br s,2H, NH₂). HRMS (FAB, MH⁺). Calculated for C₂₆H₅₄NO₇: 492.3900. Found492.3899.

Example 6

Synthesis of 1-O-hexadecyl-3-O-(p-methoxyphenyl)-sn-gylcerol (I) and2-O-hexadecyl-3-O-(p-methoxyphenyl)-sn-glycerol (II)

A mixture of 3-O-(p-methoxyohenyl)-sn-glycerol (0.737 g, 3.7 mmol) anddi-n-butyltin oxide (1.11 g, 4.46 mmol) in dry methanol (10 ml) wasrefluxed, with stirring, until the oxide was dissolved. The solvent wasevaporated, and the solid was dried under vacuum for 3 hours; the driedsolid was then dissolved in DMF (30 ml) and cesium flouride (1.5 g) and1-bromohexane (1.57 ml, 5.13 mmol) were added. The resulting mixture wasstirred at room temperature until TLC (4:1 hexane-ethyl acetate)indicated that the reaction was complete. Ethyl acetate (20 ml) andwater (0.5 ml) were then added, and the mixture was stirred for 30minutes. The resulting white solid was filtered, and the solventevaporated to give a crude mixture of I and II. This mixture ofmonoalkylated products was separated by column chromatography.

(I): 1.42 g (90%). [α]_(D) ¹H-NMR: δ6.84-6.78 (m, 4H, Ph), 4.13 (m, 1H,H-2), 4.03-3.95 (m, 2H, H-3a, H-3b), 3.76 (s, 3H, OCH₃), 3.57 and 3.54(dd, 2H, J_(1a,1b)=12 Hz, J_(1,2)=4.0 Hz, H-1a, H-1b), 3.46 (t, 2H,J=4.0 Hz, OCH₂), 2.56 (1H, OH), 1.6 (t, 2H, J=6.0 Hz, CH₂), 1.25 (s,26H, CH₂), 0.88 (t, 3H, J=6.0 Hz, CH₃). ¹³C-NMR: δ115.89, 114.99 (Ar),72.02 (OCH₂), 71.97 C-1), 70.16 (C-3), 69.53 (C-2), 56.02 (OCH₃).

(II): (76%). ¹H-NMR: δ6.87-6.80 (m, 4H, Ph), 3.89 (d, 2H, J=4.4 Hz),3.73-3.48 (m, 5H, H-1a, H-1b, H-2, H-3a, H-3b), 3.63 (s, OCH₃), 2.25(1H, OH), 1.6 (t, 2H, J=6.0 Hz, CH₂), 1.25 (s, 26H, CH₂), 0.88 (t, 3H,J=6.0 Hz, CH₃). ¹³C-NMR: δ115.93, 114.99 (Ar), 78.69 (C-2), 62.75 (C-1),56.03 (OCH₃).

Example 7

Synthesis of 1-O-hexadecyl-2-O-methyl-3-O-(p-methoxyphenyl)-sn-glycerol

General procedure for alcohol methylation: sodium hydride (2.5 mmol), indry DMF, was added portionwise at zero deg. C. to stirred alcoholsolutions (1 mmol). The resulting mixture was stirred for 30 minutes,and methyl iodide was then added (2.5 mmol). The reaction was stirred atroom temperature; once complete, methanol was added at zero deg. C. toquench excess sodium hydride. Solvent was then evaporated under vacuum,and the residue was dissolved in ethyl acetate. The organic solution waswashed with water and brine, dried (Na₂SO₄), filtered and evaporated.

1-O-Hexadecyl-3-O(p-methoxyphenyl)-sn-glycerol (1.2 g, 2.84 mmol) wasmethylated to give1-O-hexadecyl-2-O-methyl-3-O-(p-methoxyphenyl)-sn-glycerol as a whitesolid (1.2 g) in 97% after column purification. [α]_(D)−6.9 deg.;¹³C-NMR: δ115.90, 114.99 (Ar), 78.8 (C-2), 72.02, 71.98, 70.20 (C-1,C-3, OCH₂), 57.88, 56.02 (OCH₃).

Example 8

Synthesis of 2-O-acetyl-3,4,6-tri-O-benzyl-α, β-D-glucopyranosyltrichloroacetimidate

To a solution of 1,2-di-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranose (1g, 1.87 mmol) in dry DMF (10 ml) 0.213 g (2.32 mmol) of hydrazineacetate was added. This mixture was stirred under nitrogen for 4 hours.After this time, TLC (4:1 hexaneethyl acetate) showed that the reactionwas complete. The mixture was then diluted with ethyl acetate, andwashed with water and brine. The organic layer washed (sodium sulfate),filtered and then evaporated to give the crude hemiacetalquantitatively, the crude hemiacetal, pure enough to continue with, wasthen dried under vacuum for 4 hours and dissolved in dry dichloromethane(30 ml). Trichloroacetonitrile (0.231 ml) and anhydrous potassiumcarbonate (1.22 g) were added, and the resulting mixture was stirred for3 hours under nitrogen. TLC (4:1 hexane:ethyl acetate) showed traces ofthe crude hemiacetal, and the faster-running2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-glucopyranosyl trichloroacetimidate.The reaction was quenched by filtration of the inorganic base through apad of Celite 545, and the solvent was evaporated. Crude2-O-acetyl-3,4,6-tri-O-benzyl-α, β-D-glucopyranosyl trichloroacetimidatewas then purified through a short column using 8:1 hexane:ethyl acetateto give the glucosyl donor2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-glucopyranosyl trichloroacetimidatein 85% yield. ¹H-NMR: δ8.63 (s, 0.46H, NH), 8.56 (s, 0.56H, NH),7.32-7.15 (m, 15H, 3Ph), 6.52 (d, 0.53H, J_(1,2) =3.5 Hz, H-1a), 5.74(d, 0.46H, J_(1,2)=8.0 Hz, H-1_(b)), 5.29 (dd, 0.46H, J_(2,3)=9.4 Hz,H-2b isomer), 5.09 (dd 0.53H, J_(2,3)=10.0 Hz, H-2a isomer), 1.99 (s,CH₃CO).

Example 9

Synthesis of 2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-mannopyranosyltrichloroacetimidate

The benzylated 1,2-orthoester (3.0 g), prepared as described above, washydrolyzed in HOAc 80% (50 ml) at room temperature for 6 hours. Afterthis time, TLC (2:1 hexane:ethyl acetate) showed complete conversion ofthe ester into a slower moving material. Acetic acid was coevaporatedwith toluene, under vacuum, to give the pure hemiacetal quantitatively(3.0 g); the crude hemiacetal was then dried overnight, under vacuum,following which it was treated with acetonitrile-potassium carbonate togive 2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-mannopyranosyltrichloroacetimidate in 96% (3.8 g). ¹H-NMR: δ8.71 (s, NH), 8.63 (s,NH), 7.35-6.78 (m, 15H, 3Ph), 6.29 (d, H-1b), 5.89 (d, H-1a), 5.49 (dd,H-2), 4.89-4.47 (m), 4.06-3.68 (m), 2.18 (s, 3H, CH₃).

Example 10

Glycosylation of 1-O-hexadecyl-2-O-methyl-sn-glycerol with2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-glucooyranosyl trichloroacetimidateand 2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-mannopyranosyltrichloroacetimidate

A mixture of the glucosyl donor (1.4 micromolar) and1-O-hexadecyl-2-O-methyl-sn-glycerol (1.3 micromolar), prepared asdescribed above, in 30 ml of anhydrous dichloromethane was stirred underdry nitrogen, with molecular seives 3 Å for 20 minutes at roomtemperature. The mixture was cooled at −78 deg. C., and trimethylsilyltrifluoromethanesulfonate (50 micromoles. 0.035 eq.) was added. In everycase, the reaction was complete in 10 minutes. The Lewis acid wasneutralized at room temperature with triethylamine (20 microliters), thesolvent was evaporated, and the crude 1,2-trans-glycopyranosides werepurified by column chromatography.

Example 11

Synthesis of1-O-hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-β-D-glucopyranosyl)-sn-glycerol

1-O-Hexadecyl-2-O-methyl-sn-glycerol (433.3 mg, 1.3 mmol) prepared asdescribed above, was glucosylated with 2-O-acetyl-3,4,6-tri-O-benzyl-α,β-D-glucopyranosyl trichloroacetimidate (930 mg, 1.4 mmol) to give1-O-Hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-β-D-glucopyranosyl)-sn-glycerolin 76% yield (805 mg). [α]_(D) −8.5 deg. ¹H-NMR: δ7.32-7.15 (m, 15H,3PhCH₂) 4.99 (dd, 1H, J_(1′,2a′)=2.5 Hz, J_(1′,2e′)−1.0 Hz, H-1′), 3.93(m, 1H, H-5′), 3.44 (s, 3H, OCH₃), 2.15 (ddd, 1H, J_(2e′,3′)=4.7 Hz,J_(2e′,2a′)=11.5 Hz,H-2e′), 1.7 (ddd, 1H, H-2a′, 1.25 (s, 26H, CH₂),0.87 (t, 3H, CH₃). ¹³C-NMR: δ98.05 (C-1′), 79.33, (C-5′), 62.07 (C-6′),57.94 (OCH₃), 37.36 (C-2′), 31.90, 29.64, 29.48, 29.31, 26.06, 22.67(CH₂), 14.06 (CH₃).

Example 12

Synthesis of1-O-hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-α-D-mannopyranosyl)-sn-glycerol

1-O-Hexadecyl-2-O-methyl-sn-glycerol (871 mg, 2.6 micromolar) wasglucosylated with 2-O-acetyl-3′,4′,6′-tri-O-benzyl-α,β-D-mannopyranosyltrichloroacetimidate (1.90 g, 2.9 mmol), prepared as described above, togive1-O-hexadecyl-2-O-methyl-3-O-(2-O-acetyl-3′,4′,6′-tri-O-benzyl-α-D-mannopyranosyl)-sn-glycerolin 87% yield (1.83 9). [α]_(D) +37.5 deg. ¹H-NMR: δ7.33-7.12 (m, 15H,3PhCH₂), 5.37 (dd, 1H, J_(2′,3′)=2.7 Hz, H-1′), 4.86 and 4.49 (2d, 2H,J=12 Hz, CH₂Ph), 2.14 (s, 3H, CH₃CO), 1.54 (t, 2H, J=6 Hz), 1.25 (s,26H), 0.87 (t, 3H). ¹³C-NMR: δ170.39 (CO), 138.50, 132.29, 128.28,127.55 (Ph), 98.17 (C-1′), 58.07 (CH₃O), 31.90, 29.51, 26.10, 21.07(CH₂), 14.07 (CH₃).

Example 13

Deacetylation of1-O-hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-β-D-glucopyranosyl)-sn-gylceroland1-O-hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-α-D-mannopyranosyl)-sn-gylcerol

1-O-Hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-β-D-glucopyranosyl)-sn-gylceroland 1-O-hexadecyl-2-O-methyl-3-O-(2′-O-acetyl-3′,4′,6′-tri-O-benzyl-α-D-mannopyranosyl)-sn-glycerol,prepared as described above, were deacetylated at room temperature withdry ammonia gas dissolved in dry methanol in 15 minutes. This reactionwas quantitative and gave very pure products. Methanol was evaporated,and the resulting alcohols were dried under vacuum.

Example 14

Synthesis of1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2-O-methyl-β-D-glucopyranoyl)-sn-gylcerol

1-O-Hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-β-D-glucopyranosyl-sn-glycerol(149 mg), prepared as described above, was 2-O-methylated as describedabove, in 97% yield (145 mg). [α]_(D) −9.7 deg. (c 1.2, chloroform)¹³C-NMR: δ138.89, 138.34, 128.34, 127.93, 127.71, 127.55 (Ph), 103.88(C-1′), 60.45, 57.88 (CH₃O), 31.95, 29.69, 29.52, 29.35, 26.16, 22.68(CH₂), 14.06 (CH3).

Example 15

Synthesis of1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2-O-methyl-α-D-mannopyranosyl)-sn-gylcerol

1-O-Hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-α-D-mannopyranosyl-sn-gylcerol (39.6 mg), prepared as described above, was 2-O-methylatedas described above, in 98% yield (39 mg). [α]_(D) +38.3 deg. ¹³C-NMR:δ138.89, 138.34, 128.34, 127.93, 127.71, 127.55 (Ph), 98.5 (C-1′),59.99, 57.80 (CH₃O), 31.95, 29.69, 29.52, 29.35, 26.16, 22.68 (CH₂),14.06 (CH₃).

Example 16

Xanthates

Synthesis

Sodium hydride (15 mg. 0.62 micromolar) was added to an ice-coldsolution of alcohol (150 mg, 0.32 micromolar) and imidazole (4 mg, 0.55micromolar) in dry THF (5 ml). The mixture was stirred for 1 hour atroom temperature under dry nitrogen, and carbon disulfide (0.32micromolar) was then added. Stirring was continued for 20 minutes, andmethyl iodide (2.5 micromolar) was then added. The reaction wasmonitored by TLC (3:1 hexane:ethyl acetate) and it showed in every casecomplete conversion of the respective alcohols into xanthated compounds.Methanol was added at 0 deg. C. to quench the excess sodium hydride;solvent was evaporated, and the residue was dissolved in ether. Theorganic solution was washed with water, dilute hydrochloric acid andthen water; the organic layer was then dried (sodium sulfate) andevaporated.

Reduction

A solution of the resulting xanthated compound (100 mg, 0.117micromolar) in 4 ml of dry toluene was added dropwise to a refluxingsolution of tributyl tin hydride (0.31 ml, 1.17 micromolar) in 2 ml drytoluene containing α,α′-azobisisobutyronitrile (AIBN, 5 mg). Thereaction was monitored by TLC (4:1 hexane:ethyl acetate), and when itwas complete, the solvent was evaporated, and the residue was purifiedby column chromatography; the column was first eluted with hexane andthen with 20:1 hexane:ethyl acetate, 15:1 hexane:ethyl acetate, and 10:1hexane:ethyl acetate to collect pure deoxygenated1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2′-deoxy-β-D-arabinopyransoyl)-sn-glycerolor1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2′-deoxy-β-D-arabinopyransoyl)-sn-glycerol.

1-O-Hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2′-deoxy-β-D-arabinopyransoyl)-sn-gylcerol:80 mg (92%). [α]_(D) −7.1 deg. ¹H-NMR: δ7.32-7.18 (m, 15H, 3PhCH₂), 4.89(d, 1H, J=11.0 Hz, OCH₂Ph), 4.68 (d, J 11.0 Hz, OCH₂Ph), 4.63 (d, 2H, J11.0 Hz, OCH₂Ph), 4.54 (d, 2H, J=11.0 Hz, OCH₂Ph), 4.46 (dd, 1H,J_(1′,2e′)2.0 Hz, J_(1′,2a′)9.5 Hz, H-1′), 3.97 (m, 1H, H-5′), 3.73-3.26(m, 9H), 3.44 (s, OCH₃), 2.36 (ddd, 1H, J_(2e′,3′)=5.0 Hz,J_(2′e,2′a)=12.0 Hz, H-2′e), 1.73-1.43 (m, 5H), 1.25 (s, 26H, CH₂), 0.87(t, 3H, CH₃). ¹³C-NMR: δ138.46, 128.41, 128.32, 127.97 127.68, 127.51(Ph), 100.2 (C-1), 57.93 (OCH₃), 36.68 (C-2′), 31.94, 29.68, 29.52,29.35, 26.14, 22.67, 14.06 (CH₃).

1-O-Hexadecyl-2-O-methyl-3-O-(3′,4′,6′tri-O-benzyl-2′-deoxy-α-D-arabinopyransoyl)-sn-gylcerol82 mg (94%). [α]_(D) +25.5 deg. ¹H-NMR: δ7.32-7.15 (m, 15H, 3PhCH₂),4.97 (dd, 1H, H-1′), 4.89 and 4.52 (d, 2H, J=11.0 Hz, OCH₂Ph), 4.66 and4.50 (d, 2H, J 12.0 Hz, OCH₂Ph), 4.68 and 4.62 (d, 2H, J=12.0 Hz,OCH₂Ph), 3.98 (m, 1H, H-5′), 1.25 (s, 26H, CH₂), 0.87 (t, 3H, CH₃).¹³C-NMR: δ138.70, 138.59, 138.16, 128.28, 127.58, 97.82 (C-1′), 58.02(OCH₃), 35.45 (C-2′), 31.90, 29.64, 29.48, 29.31, 26.08, 22.64, 14.06(CH₃).

Example 17

Debenzylation

Protected glycosides, see above, were dissolved in 1:1 THF-HOAc, and 1-2equivalents (in weight) of palladium on charcoal were added. Thismixture was degassed under vacuum, then hydrogen was let into thereactor. This process was done three times; the mixture was then stirredat room temperature, under a balloon pressure of hydrogen. The reactionwas usually complete in 4-5 hours (TLC 10:1:0.2 ethylacetate:methanol-water). The catalyst was filtered through a pad ofCelite 545, and washed with a large volume of solvent (1:1 THF-HOAc).The solvents were evaporated under vacuum, and trace HOAc wasco-evaporated by distilling with toluene.

Deprotected glycosides were purified by column chromatography using amixture of distilled solvents (10:1 ethyl acetate-methanol). Thepurified glycosides were then filtered in distilled methanol throughlipophilic Sephadex LH-20 to remove low molecular weight impurities,such as salts.

1-O-Hexadecyl-2-O-methyl-3-O-(2′-deoxy-β-D-arabinopyransoyl)-sn-glycerol:1-O-hexadecyl-2-O-methyl-3-O-(3,4,6-tri-O-benzyl-2′-deoxy-β-D-arabinopyranosyl)-sn-glycerol(30 mg) was debenzylated to give this compound in 96% yield (14 mg).[α]_(D) −14.7 deg. ¹³C-NMR: δ100.3 (C-1′), 78.7 (C-5′), 62.05 (C-6),58.0 (OCH₃) 38.5 (C-2′), 31.95, 29.69, 29.35, 26.11, 22.62 (CH₂), 14.09(CH₃).1-O-Hexadecyl-2-O-methyl-3-O-(2′-deoxy-α-D-arabinopyransoyl)-sn-glycerol:1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2′-deoxy-α-D-arabinopyranosyl)-sn-glycerol(35 mg) gives the debenzylated compound (21 mg) in 94% yield [α]_(D)+45.0 deg. ¹H-NMR: δ4.9 (dd, 1H, J_(1′2a′)=2.5 Hz, J_(1′2e′)−1.0 Hz,H-1′), 3.93 (m, 1H, H-5′), 3.44 (s, 3H, OCH₃), 2.15 (ddd, 1H,J_(2e′3′)=4.7 Hz, J_(2e′,2a′)=11.5 Hz, H-2e′), 1.7 (ddd, 1H, H-2a′, 1.25(s, 26H, CH₂), 0.87 (t, 3H, CH₃). ¹³C-NMR: δ98.05 (C-1′), 79.33, (C-5′),62.07 (C-6′), 57.94 (OCH₃), 37.36 (C-2′), 31.90, 29.64, 29.48, 29.31,26.06, 22.67 (CH₂), 14.06 (CH₃).1-O-Hexadecyl-2-O-methyl-3-O-(2′-methyl-β-D-glucopyransoyl)-sn-glycerol:142.1 mg of1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2′-O-methyl-β-D-glucopyransoyl)-sn-glycerolwas debenzylated in 96% yield (89 mg) to give the debenzylated form inas a white, amorphous solid. [α]_(D) −14.7 deg. ¹³C-NMR: δ103.64 (C-1′),62.36 (C-6′), 60.61, 57.89 (OCH₃), 31.90, 29.64, 29.48, 29.31, 26.06,22.67 (CH₂), 14.06 (CH₃).1-O-Hexadecyl-2-O-methyl-3-O-(2′-O-methyl-α-D-mannopyransoyl)-sn-glycerol:1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-2-O-methyl-α-D-mannopyransoyl)-sn-glycerol(39 mg) was debenzylated to afford a white amorphous solid (25 mg, 94%).[α]_(D) +40.0 deg. ¹³C-NMR: δ98.89 (C-1′), 79.5 (C-5′), 62.13 (C-6′),60.1, 58.10 (OCH₃), 31.90, 29.64, 29.48, 29.31, 26.06, 22.67 (CH₂),14.06 (CH₃).1-O-Hexadecyl-2-O-methyl-3-O-(α-D-mannopyransoyl)-sn-glycerol:1-O-hexadecyl-2-O-methyl-3-O-(3′,4′,6′-tri-O-benzyl-α-D-mannopranosyl)-sn-glycerol(40 mg) was debenzylated to give a white amorphous solid in 95% yield(25 mg). %). [α]_(D) +57.2 deg. ¹H-NMR: δ4.93 (d, 1H, J_(1′2′)=1.8 Hz,H-1), 4.10 (m, 1H, H-5′), 1.25 (s, 26H, CH₂), 0.87 (t, 3H, CH₃).

Example 18

Antiproliferative Effects

The effect of1-O-hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-β-D-O-glucopyranosyl)-sn-glycerolon the proliferation of MCF-7 breast carcinoma, A549 lung carcinoma, T84breast carcinoma and A427 colon carcinoma cell lines after 72 h isdisplayed below (see Table 1). Concentrations that inhibited cell growthby 50% (Gl₅₀) in comparison to control (untreated) cultures were 9, 17,24.5 and >30 micromolar for A549, MCF-7, A427 and T84 cell lines,respectively.

TABLE 1 Lipid Concentration Cell Type (μM) MCF-7 T84 A549 A427  0 100100 100  100  5 81 ± 8 96 ± 10 91 ± 9  93 ± 12 10 71 ± 6 79 ± 12 45 ± 392 ± 6 15 58 ± 5 77 ± 13 15 ± 2  86 ± 10 20 38 ± 2 79 ± 16 0 71 ± 8 30 6 ± 3 61 ± 13 0 24 ± 4

Table 2 (see below) shows the effect of1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-βD-glucopyranosyl)-sn-glycerolon the proliferation of the cell lines growing in 10% FBS-supplementedmedium. The Gl₅₀ values were 6.5, 7, 8.3 and 12.2 micromolar for MCF-7,A427, A549 and T84 cells, respectively. Concentrations of1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-D-glucopyranosyl)-sn-glycerolcytotoxic to the cells were determined to be 10.5 micromolar for bothA549 and A427 cells, 16 micromolar for MCF-7 and 20 micromolar for T84cells.

TABLE 2 Lipid Concen- tration (μM) MCF-7 A549 A427 T84 0 100  100  100 100  5 60.60 ± 7.14 82.28 ± 4.66 68.98 ± 9.25 89.71 ± 12.72 6.5  44.05 ±11.70 67.39 ± 6.18 49.74 ± 5.16 — 7.5 — — — 75.16 ± 10.79 8.0 31.46 ±5.56  45.17 ± 12.06  4.29 ± 5.16 — 10.0 14.42 ± 8.39 0 0 64.89 ± 11.5612.5  3.90 ± 5.75 0 0 39.59 ± 9.50  15.0 0 0 0 15.06 ± 7.23  20.0 0 0 00

The effect of1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-D-glucopyranosyl-sn-glycerol(A) and of of1-O-hexadecyl-2-O-methyl-3-O-(2′-acetamido-2′-deoxy-β-D-glucopyranosyl)-sn-glycerol(B) on the growth of the ovarian cancer cell line OVCAR-3 was comparedwith that of edelfosine (ET-18-O-CH₃; C), miltefosine(hexadecylphosphocholine, D), and erucylphosphocholine (E) (see Table 3,below). The Gl₅₀ value for the glycolipids were 12 micromolar for A and4 micromolar for B, while for the phospholipids, Gl₅₀'s were 24micromolar for C and >30 micromolar for D and E.

TABLE 3 Lipid Concentration (μM) A B C D E  0 100 100 100 100 100  542.9 ± 13.4 68.8 ± 5.8 105.8 ± 8.5  93.8 ± 16.5 104.0 ± 12.2 10 7.7 ±2.3 52.9 ± 3.9 77.5 ± 1.0 90.3 ± 11.3 102.3 ± 16.1 15  0 34.6 ± 7.3 70.7± 9.9 67.4 ± 14.5 98.6 ± 7.8 20 —  26.0 ± 10.9  68.7 ± 13.6 62.3 ± 10.790.2 ± 6.6 30 —  4.7 ± 4.8 20.4 ± 8.1 67.2 ± 12.5   60 ± 10.7

The Gl₅₀ values for edelfosine (1), 2′-deoxy-β-D-arabinopyranosyl (2),2′-deoxy-α-D-arabinopyranosyl (3), 2-O-methyl-β-D-glucopyranosyl (4),2′-O-methyl-β-D-mannopyranosyl (5) and α-D-mannopyransoyl (6) on A-549,MCF-7, Lewis Lung, MCF-7/Adr (adriamycin-resistant), P388, P-388/Adr,L1210 and L1210/vmdr cells were determined and are set forth below (seeTable 4) as the concentration (micromolar) of lipid required to inhibitthe growth of fifty percent of the cells in culture.

TABLE 4 Cell Line 1 2 3 4 5 6 A549 5.05 ± 0.80^(a)  9.90 ± 0.99 19.65 ±0.07 18.30 ± 0.14 15.55 ± 0.07 18.10 ± 0.14 MCF7 9.66 ± 2.50^(a)  6.93 ±0.12 24.45 ± 0.64 23.05 ± 0.64 18.20 ± 0.00 21.70 ± 0.57 MCF7/adr 30.35± 5.07^(b) 12.85 ± 0.85 24.40 ± 0.42 21.75 ± 0.63 18.55 ± 0.07 23.30 ±0.71 HT29 2.20 ± 0.27^(b)  7.59 ± 0.23 29.60 ± 0.28 — 20.00 ± 0.28 23.20± 0.85 Lewis Lung 30.24 ± 6.32^(c) 11.05 ± 0.49 — 26.00 ± 0.71 — — P3884.33 ± 1.37^(d) 12.65 ± 0.78 — 18.10 ± 1.13 — — P388/adr 6.39 ± 2.43^(d)10.25 ± 0.34 — 29.30 ± 5.66 — — L1210 3.32 ± 1.68^(c)  7.02 ± 0.49 18.75± 0.49 — 15.45 ± 0.35 16.20 ± 0.99 L1210/ 10.99 ± 6.36^(c)  7.09 ± 0.3326.95 ± 1.06 — 16.50 ± 1.13 18.90 ± 0.00 vmdr

Gl₅₀ values are given as the mean ± standard deviation; the Gl₅₀ valuefrom each experiment “n” was generated from three individual wells ontwo separate plates (six total wells); n=1 for compounds 3-6; n=2 formost cell lines treated with compound 2 ^(a)n=3; ^(b)n=2; ^(c)n=6;^(d)n=8.

What is claimed is:
 1. An etherlipid having the formula:

wherein: R¹ is a group having the formula —Y¹Y²; Y¹ is a group havingthe formula—(CH₂)_(n1)(CH═CH)_(n2)(CH₂)_(n3)(CH═CH)_(n4)(CH₂)_(n5)(CH═CH)_(n6)(CH₂)_(n7)(CH═CH)_(n8)(CH₂)_(n9)and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 3 to23, n1 is equal to zero or an integer of from 1 to 23, n3 is equal tozero or an integer of from 1 to 20, n5 is equal to zero or an integer offrom 1 to 17, n7 is equal to zero or an integer of from 1 to 14, n9 isequal to zero or an integer of from 1 to 11, and each of n2, n4, n6 andn8 is independently zero or 1; Y² is CH₃, CO₂H or OH; R² is O, S, NH,—OC(O)— or NHCO; R³ is a group having the formula

wherein: X¹ is O or S; X² is H, OH, NH₂, NHCH₃, N(CH₃)₂, OCH₃, NHCOCH₃,F or Cl; X³ is H, OH, NH₂, NHCH₃, N(CH₃)₂, OCH₃, F or Cl, provided thatwhen X² is H, X³ is H, NH₂, NHCH₃, OCH₃, F or Cl; X⁴ is H, OH, OPO₃ ³⁻or OSO₃ ²⁻ and X⁵ is H, OH, NH₂, NHCH₃ or N(CH₃)₂; each of X⁶ and X⁷ isH, OH, —OX⁹, OPO₃ ³⁻ or OSO₃ ²⁻; X⁸ is CH₂OH when the sugar isunmodified at the C-6 position, or a group having the formula COOX¹⁰,when the sugar is modified at this position; X⁹ is a tetrose, pentose,hexose or heptose sugar; X¹⁰ is H, CH₃ or a group having the formulaY¹Y²; and wherein no more than two of X², X³, X⁴, X⁵, X⁶ and X⁷ are OHand no more than two of X²/X³, X⁴/X⁵ and X⁶/X⁷ are H/OH or OH/H when X⁸is CH₂OH.
 2. The etherlipid of claim 1, wherein R¹ is a group having theformula Y¹CH₃.
 3. The etherlipid of claim 2, wherein R¹ is a grouphaving the formula (CH₂)_(n1)CH₃.
 4. The etherlipid of claim 3, whereinR¹ is (CH₂)₁₅CH₃ or (CH₂)₁₇CH₃.
 5. The etherlipid of claim 1, wherein R²is O.
 6. The etherlipid of claim 1, wherein R³ is the alpha anomericform.
 7. The etherlipid of claim 1, wherein R³ is the beta anomericform.
 8. The etherlipid of claim 1, wherein X¹ is O.
 9. The etherlipidof claim 1, wherein X⁴ is OH, X⁵ is H, X⁶ is H, X⁷ is OH and wherein X²is H and X³ is NH₂, H or OCH₃ or wherein X³ is H and X² is OH, H orOCH₃.
 10. The etherlipid of claim 1, wherein R¹ is (CH₂)₁₅CH₃, R² is Oand X¹ is O.
 11. The etherlipid of claim 10, wherein X⁴ is OH, X⁵ is H,X⁶ is H, X⁷ is OH and wherein X² is H and X³ is NH₂, H or OCH₃ orwherein X³ is H and X² is OH, H or OCH₃.
 12. The etherlipid of claim 10,wherein X² is H, X³ is OH, X⁴ is OH, X⁵ is H, X⁶ is H, X⁷ is OH and X⁸is COOX¹⁰ and wherein X¹⁰ is CH₃ or a group having the formula Y¹Y². 13.A pharmaceutical composition comprising the etherlipid of claim 1 and apharmaceutically acceptable medium.
 14. The composition of claim 13,wherein the pharmaceutically acceptable medium comprises a lipid carrierand wherein the etherlipid is associated with the carrier.
 15. Thecomposition of claim 14, wherein the lipid carrier is a fatty acid,phospholipid, micelle, lipid complex, liposome or lipoprotein.
 16. Amethod of treating an animal afflicted with a cancer which compriseadministering an anticancer-effective amount of the composition of claim13 to the animal.
 17. The method of treating an animal as described inclaim 16, wherein the composition comprises a liposome.
 18. The methodof treating an animal as described in claim 16, further comprisingadministering an additional bioactive agent to the animal.
 19. Theetherlipid of claim 1, is1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-D-glucopyranosyl)-sn-glycerol.